HCMV US28 regulation of host cell signaling in viral latency and reactivation

HCMV US28 在病毒潜伏期和再激活过程中对宿主细胞信号传导的调节

• 批准号: 10629177
• 负责人: DANIEL N STREBLOW
• 金额: $ 39.43万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10629177
• 关键词: Affect; Allogenic; Binding; Bone Marrow Transplantation; C-terminal; CCL2 gene; CD34 gene; CX3C Chemokines; Cell Differentiation process; Cell Lineage; Cell Maintenance; Cells; Cercopithecine Herpesvirus 1; Collaborations; Couples; Cytomegalovirus; Data; Development; Dissection; Epidermal Growth Factor Receptor; Fractalkine; Genes; Goals; Graft Rejection; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Human; In Vitro; Infection; Ligand Binding; Ligands; Link; MEKs; Mediating; MicroRNAs; Molecular; Morbidity - disease rate; Mutation; Mutation Analysis; Myelogenous; Myelosuppression; N-terminal; Organ; Organ Transplantation; PIK3CG gene; Pathway interactions; Play; Point Mutation; Population; Process; Productivity; Proliferating; Proteins; RANTES; Receptor Signaling; Recombinant Delta Chemokine; Regulation; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Solid; Stem cell transplant; System; Tail; Time; Transplant Recipients; Undifferentiated; Viral; Viral Proteins; Virus; Virus Latency; beta-Chemokines; chemokine; chemokine receptor; functional outcomes; gene product; graft failure; humanized mouse; in vitro Model; in vivo; in vivo Model; inhibitor; knock-down; member; monocyte; mortality; mutant; neutralizing antibody; novel therapeutics; prevent; programs; reactivation from latency; rho; sensor; sensor technology

SUMMARY - PROJECT 3 Human cytomegalovirus (HCMV) is a b-herpesvirus infecting 44-100% of the population and remains a significant cause of morbidity and mortality in solid organ transplant (SOT) and allogeneic hematopoietic stem cell transplant (SCT) recipients. Infection in SCT patients is often associated with myelosuppression and graft failure due to virus reactivation from latency, but the associated mechanisms are still largely unknown. HCMV encodes multiple latency-associated gene products including the chemokine receptor US28, which binds CC- chemokines as well as the CX3C-chemokine Fractalkine. We have demonstrated that US28 ligand-dependent signaling is required to maintain viral latency in vitro in CD34+ hematopoietic progenitor cells (HPCs) and in vivo in humanized mice. US28 was required in both systems for reactivation of latent HCMV and promotes CD34+ HPC differentiation into myeloid lineage cells, indicating that US28 plays a dynamic role in both processes. While we have identified that ligand binding activity for US28 is required for HCMV latency, we do not yet know whether this effect is ligand-specific. In addition, we have recently demonstrated that HCMV-encoded chemokine ligands modulate US28 signaling. We hypothesize that both the host CC chemokines and viral chemokines UL146/UL147 promote latency by activating specific signaling pathways while other US28 ligands help direct reactivation by modifying signaling. We hypothesize that US28 ligand binding activity (ligand-specific) is required to maintain latency by interfacing with EGFR signaling and during reactivation US28 antagonizes EGFR signaling and shifts to activating RhoA pathways to enhance productive replication. In Specific Aim 1, we will determine which US28 ligands reprogram cells during latency and reactivation using neutralizing antibodies against host and viral chemokines, viral mutants that impact the ability of US28 to selectively bind ligands, and viral mutants lacking the viral chemokines. In SA2, we will utilize our US28 interactome data generated using the BirA-Turbo proximity sensor technology to identify signaling factors that play a role in latency and reactivation. Through mutational analyses we have identified specific regions of US28, in the C’terminal tail and intracellular loop 3 domains, that modify US28 signaling. We will determine what effect these mutations have on both the US28 interactome and latency and reactivation using in vitro and in vivo models. Lastly, in SA3, we will define how US28 signaling influences other HCMV proteins (Projects 1 and 4) and miRNAs (Project 2) with specific focus on how US28 intersects with the EGFR pathway, to contribute to the combined HCMV-manipulation of EGFR signaling (Project 5) and therefore control of latency and reactivation within the overarching goals of this proposal. Results of this study will generate new virus latency and reactivation paradigms promoting the development of novel therapies to prevent virus reactivation.

摘要--项目3 人类巨细胞病毒(Hcmv)是一种b型疱疹病毒，感染44%-100%的人，至今仍是一种 实体器官移植和异基因造血干细胞致病和死亡的重要原因 细胞移植(SCT)接受者。SCT患者的感染通常与骨髓抑制和移植物有关。 失败是由于病毒从潜伏期重新激活，但相关机制在很大程度上仍不清楚。巨细胞病毒 编码多种潜伏期相关基因产物，包括趋化因子受体US28，它与CC- 趋化因子以及CX3C-趋化因子Fractalkine。我们已经证明了US28配体依赖 CD34+造血祖细胞在体外和体内维持病毒潜伏期所需的信号 在人源化的小鼠身上。在两个系统中都需要US28来重新激活潜伏的HCMV并促进CD34+ HPC分化为髓系细胞，表明US28在这两个过程中都发挥了动态作用。而当 我们已经确定了US28的配体结合活性是HCMV潜伏期所必需的，我们还不知道是否 这种效应是特定于配体的。此外，我们最近还证明了巨细胞病毒编码的趋化因子配体 调制US28信令。我们假设宿主CC趋化因子和病毒趋化因子 UL146/UL147通过激活特定的信号通路来延长潜伏期，而其他US28配体则帮助引导 通过修改信令重新激活。我们假设US28配体结合活性(配体特异性)是必需的 通过与EGFR信号交互来维持潜伏期，并在重新激活期间US28拮抗EGFR信号 并转变为激活RhoA途径以增强生产性复制。在具体目标1中，我们将确定 哪些US28配体在潜伏和重新激活期间使用针对宿主的中和抗体重新编程细胞 以及病毒趋化因子、影响US28选择性结合配体能力的病毒突变体和病毒突变体 缺乏病毒趋化因子。在SA2中，我们将利用使用Bira-Turbo生成的US28 Interactome数据 接近感应器技术，以识别在延迟和重新激活中起作用的信号因素。穿过 突变分析表明，我们已经确定了US28在C‘末端尾部和细胞内环3中的特定区域 域，修改US28信令。我们将确定这些突变对US28和US28 使用体外和体内模型的交互作用组以及潜伏期和再激活。最后，在SA3中，我们将定义 US28信号以特定的焦点影响其他HCMV蛋白(方案1和4)和miRNAs(方案2) US28如何与EGFR途径相交，促进HCMV对EGFR的联合操纵 信令(项目5)，因此在总体目标内控制延迟和重新激活 求婚。这项研究的结果将产生新的病毒潜伏和重新激活范例，促进 开发新的治疗方法以防止病毒重新激活。